Three different poly(amino acid)s are known to occur naturally: poly(ε-L-lysine) (ε-PL), poly(γ-glutamic acid) (γ-PGA), and cyanophycin (CGP). Poly(amino acid)s are present in many environments and fulfil different functions for the producing organisms (Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)). For example, Cyanophycin (multi-L-arginyl-poly-[L-aspartic acid]), which is known also as Cyanophycin Granule olypeptide (CGP), which was discovered in cyanobacteria more than 100 years ago (Borzi, A., Malpighia 1:28-74 (1887)) provides the organism with nitrogen, carbon and energy. It contains five nitrogen atoms in every building block and thereby represents an ideal intracellular nitrogen reserve (Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990)). The biocompatibility and complete biodegradability of poly(amino acid)s make them ideal candidates for many applications in human life in the fields of biomedicine, agriculture, agrochemistry, personal care, and pharmacy (Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)).
Several species of cyanobacteria including the blue green algae Spirulina have been promoted as nutritional sources for humans and animals (Kihlberg, R., A. Rev. Microbiol. 26:427-466 (1972)). CGP itself was discovered in 1887 in cyanobacteria. Most genera of cyanobacteria harbor a functional cyanophycin synthetase gene (cphA) and synthesize CGP (Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990)). Genes coding for CphA were identified also in heterotrophic bacteria (Krehenbrink, M. et al., Arch. Microbiol. 177:371-380 (2002); Fuser, G. et al., Macromol. Biosci. 7:278-296 (2007)). The branched polymer occurs in the cytoplasm as insoluble intracellular membraneless granules (Allen, M. M. et al., J. Bacteriol. 154:1480-1484 (1983)). It consists of equimolar amounts of arginine and aspartate arranged in the form of poly(aspartic acid) (PAA) backbone, with arginine moieties linked to the β-carboxyl group of each aspartic acid by its α-amino group (Simon, R. D. et al., Biochim. Biophys. Acta 420:165-176 (1976)). For large scale production, cyanobacterial cphA genes were heterologously cloned in Escherichia coli, Corynebacterium glutamicum, Ralstonia eutropha and Pseudomonas putida. CGP from recombinant bacteria contains a little lysine (Voss, I. et al., Metabol. Eng. 8:66-78 (2006)). CGP is widely spread in different natural habitats and is degraded by intracellular or by extracellular CGPases (CphB, CphE, respectively). Bacteria possessing CphE were found in various habitats; CphEPa and CphEBm were isolated and characterized from P. angulliseptica BI and B. megaterium BAC19, respectively (Obst, M. et al., J. Biol. Chem. 277:25096-25105 (2002); Obst, M. et al., Biomacromolecules 5:153-161 (2004)). CGP degradation occurs also in anaerobic habitats by strictly or facultative anaerobic bacteria such as Sedimentibacter hongkongensis KI or P. alcaligenes DIP1, respectively (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652 (2005); Sallam, A. et al., Submitted for publication (2008)). All known CGPases produced water soluble β-dipeptides from CGP which are then transported into the cells to be further catabolized (Sallam, A. et al., Submitted for publication (2008)).
Protein digestion and transport is essential for life. In ruminants for instance, the major part of dietary protein is degraded by the rumen flora to amino acids and peptides. Amino acids are incorporated into microbial protein or passed to next parts of the digestive tract or absorbed directly across the rumen wall into the blood (Faix, {hacek over (S)}. et al., Acta Vet. Brno. 70:243-246 (2001)). However, Tri- and dipeptides are more efficiently utilized than free amino acids, have greater nutritional value, are better absorbed [up to 185% greater than free amino acids (Adibi, S. A., J. Clin. Invest. 50:2266-2275 (1971)) and retain more nitrogen than intact protein contributing to enhance weight gain (Dock, D. B. et al., Biocell 28:143-150 (2004)). Absorption studies in patients with genetically impaired transport of certain amino acids showed normal absorption of these amino acids if administered as dipeptides. This indicated the presence of specialized and effective transport systems for dipeptides (Adibi, S. A., Gastroenterology 113:332-340 (1997)). Therefore, hydrolyzed protein diets are frequently applied as feed additives to recovery malnourished cases (Dock, D. B. et al., Biocell 28:143-150 (2004)).
The semi-essential amino acid arginine plays several pivotal roles in cellular physiology, and thus is applied in therapeutic regimens for many cardiovascular, genitourinary, gastrointestinal, or immune disorders (for review see (Appleton, 3., Altem. Med. Rev. 7:512-522 (2002)). The essential amino acid lysine is known as food additive for human and animal, has antiviral activity against Herpes simplex virus, and improves calcium absorption in the small intestine, and hence acts against osteoporosis (Cynober, L. A., Metabolic and therapeutic aspects of amino acids in clinical nutrition. 2nd ed. CRC Press LLC, Boca Raton, USA (2003)). The non-essential amino acid aspartate serves among others as a precursor for L-arginine, for energy metabolism (Voet, D. et al., Biochemistry. 3th ed. John Wiley and Sons Inc., New York (2004)), and is used in drug delivery for cations or for other amino acids (Cynober, L. A., Metabolic and therapeutic aspects of amino acids in clinical nutrition. 2nd ed. CRC Press LLC, Boca Raton, USA (2003)). Because amino acids have higher bioavailability in the dipeptide form, their administration as dipeptides was clinically approved and are available in market products (Duruy, A. et al., Vie. Med. Int. 9:1589 (1965); Duruy, A., Med. Int. 1:203 (1966); Sellier, 3., Rev. Med. Toulouse 5:879 (1979); De-Aloysio, D. et al., Acta Eur. Fertil. 13:133-167 (1982); Rohdewald, P., Int. J. Clin. Pharmacol. Ther. 40:158-168 (2002); Lamm, S. et al., Eur. Bull. Drug Res. 11:29-37 (2003)).
Until now, no direct applications are known for CGP itself. Previous studies on CGP were motivated by CGP as a potential source for a biodegradable PAA (Mooibroek, H. et al., Appl. Microbiol. Biotechnol. 77:257-267 (2007)). The latter has many potential applications (Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)) as a component in dialysis membranes, artificial skin and orthopedic implants or as drug carrier. PAA could also substitute non-biodegradable polyacrylates for which many technical applications are described. This is the first study on the biodegradation of CGP by mammalian, avian and fish gut flora, and subsequently on the potential applications of CGP and the dipeptides thereof as nutritional and/or therapeutic additives.
Several samples of mammalian, avian and fish gut flora were investigated for cyanophycin degradation. All samples achieved complete anaerobic CGP degradation over incubation periods of 12-48 h at 37° C. CGP degrading bacteria were found in all samples and were highly concentrated in cecum flora from rabbit and sheep and digestive tract flora from carp fish. A total of 62 axenic cultures were isolated and degraded CGP aerobically, 46 thereof degraded CGP also anaerobically over incubation periods ranging from 24 h to 7 days. HPLC analysis revealed that all isolates degraded CGP to its constituting dipeptides. Eight strains were identified by 16S rDNA sequencing and were affiliated to the genera Bacillus, Brevibacillus, Pseudomonas, Streptomyces and Micromonospora. CGP could be found in three different Spirulina platensis commercial products which contained 0.06-0.15% (wt/wt) CGP. It was now found that CGP can be degraded extracellularly CGP degradation, as well as the first evidence on CGP biodegradability in the digestive tract, and subsequently, the potential application of CGP and its dipeptides in nutrition and therapy as highly bioavailable sources for arginine, lysine, aspartate and possibly other amino acids.
CGP accumulates in cyanobacteria during the transition from the exponential to the stationary growth phase (Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990); Sherman, D. M. et al., J. Phycol. 36:932-941 (2000)). Most genera of cyanobacteria harbor a functional cyanophycin synthetase gene (cphA) and synthesize CGP (Simon, R. D. 1987. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies, pp. 199-225. In P. Fay and C. van Baalen (ed.), The Cyanobacteria, Elsevier, Amsterdam, The Netherlands; Allen, M. M. et al., Methods Enzymol. 167:207-213 (1988); Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990); Liotenberg, S. et al., Microbiology 142:611-622 (1996); Wingard, L. L. et al., Appl. Environ. Microbiol. 68:1772-1777 (2002)). cphA genes were also identified in heterotrophic bacteria (Krehenbrink, M. et al., Arch. Microbiol. 177:371-380 (2002); Ziegler, K. et al., Naturforsch. 57c:522-529 (2002)). The polymer occurs in the cytoplasm as membraneless granules and is insoluble at neutral pH as well as in physiological ionic strength (Allen, M. M. et al., J. Bacteriol. 141:687-693 (1980)). CGP accumulates under limiting conditions including low temperature, low light intensity, phosphorous or sulfur limitation (Stephan et al., Z. Naturforsch. 55:927-942 (2000)). In cyanobacteria, the molecular mass of the polymer strands range from 25 to 100 kDa (Simon, R. D., Biochim. Biophys. Acta 422:407-418 (1976)), while those from recombinant strains exhibit a lower range (25 to 30 kDa) and polydispersity. Furthermore, it was found that the polymer from recombinant strains contained lysine as an additional amino acid constituent (Ziegler, K. et al., Eur. J. Biochem. 254:154-159 (1998); Aboulmagd, E. et al., Biomacromolecules 2:1338-1342 (2001)). CGP functions as a temporary nitrogen, energy and possibly carbon reserve (Li, H. et al., Arch. Microbiol. 176:9-18 (2001); Elbahloul, Y. et al., Appl. Environ. Microbiol. 71:7759-7767 (2005)). Because CGP contains five nitrogen atoms in every building block, it fulfills the criteria for the perfect intracellular nitrogen reserve (Simon, R. D. 1987. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies, pp. 199-225. In P. Fay and C. van Baalen (ed.), The Cyanobacteria, Elsevier, Amsterdam, The Netherlands).
The intracellular degradation of CGP is catalyzed by highly specific cyanophycinases (CphB) occurring in the cytoplasm and proceeds via an α-cleavage mechanism resulting in the formation of β-dipeptides (Richter, R. et al., Eur. J. Biochem. 263:163-169 (1999)). CGP represents a valuable substrate also for bacteria not capable of CGP accumulation (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652 (2005); Sallam, A., and A. Steinbüchel. 2007a). Clostridium sulfatireducens sp. nov., a new mesophilic, proteolytic bacterium isolated from a pond sediment, able to reduce thiosulfate, sulfur and transiently sulfate), many of such bacteria were shown to possess extracellular cyanophycinases that degrades CGP to its utilizable dipeptides, which can be transported into the cell and further utilized (Sallam, A., and A. Steinbüchel. 2007b. Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1-Role of other three co-isolates in the mixed bacterial consortium. Submitted for publishing) Several examples of these enzymes were isolated and characterized, such as CphEPa from the Gram-negative bacterium Pseudomonas angulliseptica strain BI. This extracellular enzyme exhibited, similar to CphB, an α-cleavage mechanism for CGP degradation (Obst, M. et al., J. Biol. Chem. 277:25096-25105 (2002)).
Also Gram-positive bacteria were found to excrete CGPases when the extracellular CphEBm was isolated from Bacillus megaterium strain BAC19 (Obst, M. et al., Biomacromolecules 5:153-161 (2004)), both CphEPa and CphEBm were identified as serine-type hydrolases. Recent studies revealed that extracellular CGP degradation can be catalyzed also by CGPases from strict as well as facultative anaerobic bacteria, such as Sedimentibacter hongkongensis strain KI (Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652 (2005)) and Pseudomonas alcaligenes strain DIP1 (Sallam, A., and A. Steinbüchel. 2007b. Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1-Role of other three co-isolates in the mixed bacterial consortium. Submitted for publishing), respectively. All investigated CGPases yielded β-Asp-Arg dipeptides as cleavage products, however, (Asp-Arg)2 tetrapeptides were additionally detected in case of CphEBm (Obst, M. et al., Biomacromolecules 5:153-161 (2004)).
Until recently, no practical applications were known for CGP itself or for the dipeptides thereof. On contrast, economically important applications have been established for poly(aspartic acid) (PAA), which is a structural element (polymer backbone) of CGP, as a substitute for non-biodegradable polyacrylates (Schwamborn, M., Polym. Degrad. Stab. 59:39-45 (1998)). PAA can be also employed in many fields including paper, paint and oil industries (reviewed by Joentgen, W. et al. 2003. Polyaspartic acids. pp. 175-199. In: S. R. Fahnestock and A. Steinbüchel (ed.), Biopolymers, vol 7. Wiley, Weinheim). Biomedical applications have also been described for PAA (Leopold, C. S. et al., J. Pharmacokinet. Biopharm. 4:397-406 (1995); Yokoyama, M. et al., Cancer Res. 6:1693-1700 (1990)). Only recently, biomedical applications for CGP-dipeptides and possibly for CGP itself were revealed, these applications depend in first place on the astonishing wide spread of CGP-degrading bacteria in numerous investigated mammalian, avian, and fish flora, this indicated that CGP is probably degradable within the respective digestive tracts, On the other hand, the elevated bioavailability of amino acids if administrated in the dipeptide or tripeptide form is a well known theory and is effectively applied in several therapeutic fields. Thus, CGP and/or its β-dipeptides can be considered as potential natural food and/or therapeutic additives for the near future (Sallam, A., and A. Steinbüchel. 2007c. Potential of cyanophycin and its β-dipeptides as possible additives in therapy, food and feed industries).
The production and efficient isolation of CGP in semi-technical amounts was established only during the last few years. Several bacterial strains of E. coli, Ralstonia eutropha, Pseudomonas putida and Acinetobacter baylyi strain ADP1 were applied, the later showed the maximum CGP yield of about 46% (wt/wt) (Obst, M. et al., pp. 167-194. In J. M. Shively (ed.), Inclusions in Prokaryotes, vol. 1. Springer-Verlag, Berlin, Heidelberg (2006)). However, the required substrates and cultivation conditions are also crucial factors for choosing the economically appropriate CGP-producer.
It was now found that pure CGP-dipeptides can be prepared in an economical large scale process which starts from CGP-containing biomass and ends with pure CGP-dipeptides. Because strain P. alcaligenes DIP1 could show high enzyme productivity on simple growth requirements. This strain was found ideal for such a technical process.
Cyanophycin contains five nitrogen atoms in every building block and therefore accomplishes exactly the criteria for a perfect dynamic intracellular nitrogen reserve (Simon, R. D. 1987. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies, pp. 199-225. In P. Fay and C. van Baalen (ed.), The Cyanobacteria, Elsevier, Amsterdam, The Netherlands); its amount fluctuates according to the needs of the cells (Carr, N. G. 1988. Nitrogen reserves and dynamic reservoirs in cyanobacteria, p. 13-21. In L. J. Rogers and J. R. Gallon (ed.), Biochemistry of the algae and cyanobacteria, Annual Proceedings of the Phytochemical Society of Europe, Clarendon, Oxford). The polymer accumulates in cyanobacteria when the protein synthesis is diminished either naturally during the transition from the exponential to the stationary growth phase (Simon, R. D., Arch. Microbiol. 92:115-122 (1973a)) or by addition of inhibitors of protein biosynthesis (e.g. chloramphenicol) (Ingram, L. O. et al., Arch. Microbiol. 81:1-12 (1972); Simon, R. D., J. Bacteriol. 114:1213-1216 (1973b)) and the polymer disappears when balanced growth resumes (Mackerras, A. H. et al., J. Gen. Microbiol. 136:2057-2065 (1990)). CGP accumulation is also promoted by phosphorous limitation (Stephan et al., Z. Naturforsch. 55:927-942 (2000)), sulfure limitation (Ariño, X. et al., Arch. Microbiol. 163:447-453 (1995)), low temperature, low light intensity or a combination of these factors (Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)).
Different methods were developed for determination and quantification of either purified CGP or its content in cells. Arginine content of CGP was quantified colorimetrically either in hydrolyzed or in unhydrolyzed polymer by the Sakagushi reagent (Simon, R. D., J. Bacteriol. 114:1213-1216 (1973b)). The amino acid constituents of the purified cyanophycin could be determined by HPLC (Aboulmagd, E. et al., Arch. Microbiol. 174:297-306 (2000)). For rapid and sensitive determination of cyanophycin, a method based on 1H nuclear magnetic resonance (NMR) was developed (Erickson, N. A. et al., Biochim. Biophys. Acta. 1536:5-9 (2001)).
CGP degradation (intra- or extracellular) leads mainly to the release of its utilizable dipeptides, these are then split intracellularly to their constituting amino acids to be engaged into cell metabolism. Intracellular degradation of cyanophycin is catalyzed by cyanophycinases (CphB). The first cyanophycinase was described in heterocysts and vegetative cells of Anabaena cylindrica by Gupta, M. et al., J. Gen. Microbiol. 125:17-23 (1981). The enzyme is a monomeric 29.4 kDa, serine-type, and a cyanophycin-specific exopeptidase, its main degradation product was aspartate-arginine dipeptides via an α-cleavage mechanism (Richter, R. et al., Eur. J. Biochem. 263:163-169 (1999)). In the last few years, aerobic and anaerobic bacteria able to degrade cyanophycin by extracellular cyanophycinases (CphE) were isolated (Obst, M. et al., J. Biol. Chem. 277:25096-25105 (2002)); Obst, M. et al., Biomacromolecules 5:153-161 (2004); Obst, M. et al., Appl. Environ. Microbiol. 71:3642-3652 (2005); Sallam, A., and A. Steinbüchel. 2007b. Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1-Role of other three co-isolates in the mixed bacterial consortium. Submitted for publishing). Similar to CphB, the previously characterized extracellular CGPases; CphEPa and CphEBm, from Pseudomonas anguilliseptica strain B1 and Bacillus megaterium strain BAC19, respectively, were identified as serine-type, cyanophycin-specific enzymes and produced CGP-dipeptides as degradation products, however, (Asp-Arg)2 tetrapeptides were additionally detected in case of CphEBm. Labeling studies of CphEPa showed that the enzyme hydrolyses CGP at the carboxyl-terminus and successively releases n-Asp-Arg dipeptides from the degraded polymer chain end (for review see Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)). Moreover, a third extracellular cyanophycinase (CphEal) from Pseudomonas alcaligenes DIP1 was recently applied in crude form for the technical production of CGP-dipeptides (Sallam, A., and A. Steinbüchel. 2008b. Biotechnological process for the technical production of β-dipeptides from cyanophycin. Under preparation).
The production and efficient isolation of CGP in semi-technical amounts were established only during the last few years. Several bacterial strains of Escherichia coli, Ralstonia eutropha, Pseudomonas putida and Acinetobacter baylyi were successfully applied (Obst, M. et al., Biomacromolecules 5:1166-1176 (2004)). However, the biotechnological relevance of CGP was based theoretically on being a source for poly(aspartic acid) which has high potential for industrial applications [e.g. for water treatment; paper and leather industries, as dispersing agent (Roweton, S. et al., J. Environ. Polym. Degrad. 5:175-181 (1997); Mooibroek, H. et al., Appl. Microbiol. Biotechnol. 77:257-267 (2007)) or as biodegradable substitute for polyacrylate (Schwamborn, M., Polym. Degrad. Stab. 59:39-45 (1998)). PAA has also potential biomedical applications as a component in dialysis membranes, artificial skin, orthopaedic implants and as drug carrier (Leopold, C. S. et al., J. Pharmacokinet. Biopharm. 4:397-406 (1995)).
As set forth above, biomedical applications for CGP-dipeptides and possibly for CGP itself were revealed, this indicated that CGP is probably degradable within the mammalian and fish digestive tracts; this represented the polymer and the dipeptides thereof as potential natural food and/or therapeutic additives for the near future. Accordingly, a large scale process for the production of dipeptides from CGP using crude CphEal from P. alcaligenes strain DIP1 was recently constructed. This original process comprised three phases; Phase I: large scale extraction and purification of CGP, Phase II: large scale production of crude CphEal powder, Phase III: degradation of CGP to its dipeptides was set up as described hereinbefore. It was now found, the latter two phases of the original process can be greatly optimized for future applications. Moreover, CphEal was technically purified from the crude powder and the biochemical characteristics thereof were revealed.